Delivery of card protein as therapy for occular inflammation

ABSTRACT

The present invention provides methods and compositions for treating and/or preventing age related macular degeneration and other conditions involving macular degeneration, ocular neovascularization, or ocular inflammation. In an exemplary embodiment, a method is disclosed that involves administering an expression vector that delivers a secretable and cell penetrating CARD to a subject in need of treatment or prevention of age-related macular degeneration or another condition involving macular degeneration or ocular neovascularization.

STATEMENT OF GOVERNMENT FUNDING

The invention was made with government support under EY020825 andEY021721 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofInternational PCT application PCT/US2014/023262, filed Mar. 11, 2014which is related to U.S. Provisional Application No. 61/776,076 filedMar. 11, 2013, to which priority is claimed under 35 USC 119, and whoseentire disclosures are incorporated herein by reference.

BACKGROUND AND SUMMARY

Dry Age-related macular degeneration (AMD) has been associated with anincrease in oxidative stress and inflammatory processes within theretina. Oxidized molecules like 4-hydroxynonenal (4-HNE) have beendetected in the eyes of dry AMD patients, supporting the role of theseprocesses in the diseases. The purpose of this work is to develop an AAVvector that delivers a secretable and cell penetrating caspaseactivation and recruitment domain (CARD) from the Apoptosis-associatedspeck-like protein containing a CARD (ASC) gene to study the role of theproinflammatory cytokine IL-1 beta (IL-1β) in dry AMD and for developingtreatments of diseases, conditions or disorders associated withoxidative stress and inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Development of a lentiviral vector delivering the fused TatCARDprotein. The caspase activation and recruitment domain (CARD) from theApoptosis-associated speck-like protein containing a CARD (ASC) gene,which is known to link the NRLP3 protein of the inflammasome to thecaspase-1 enzyme, was fused to the cell-penetrating peptide derived fromthe HIV Tat protein (Tat) by PCR. This DNA fragment was cloned into thepCDH-EF1-MCS-T2A-Puro lentiviral vector (LV) plasmid within the multiplecloning site (MCS) to create the fused gene TatCARD-T2A-PuroR. Cellstransduced with the LV/EF1-TatCARD-T2A-PuroR will express the fusedTatCARD-T2A-PuroR protein which will be separated into the TatCARD andPuroR proteins by the self-cleaving peptide T2A. Cells expressing theTatCARD fused protein can then be selected by their resistant topuromycin in vitro.

FIG. 2. DNA sequence (SEQ ID NO: 2) and protein translation (SEQ IDNO: 1) of the fusion protein TatCARD. The sequence highlighted in darkgray corresponds to the sequence of the Tat peptide, while the sequencehighlighted in light gray corresponds to the CARD domain. The predictedmolecular weight (MW) of the TatCARD fused protein is 11.8 KDa based onits amino acid sequence.

FIG. 3. The CARD domain of the ASC gene binds to caspase-1. Proteinlysate from ARPE-19 transduced with lentiviral vector delivering eitherthe puromycin resistance gene (PuroR) or the TatCARD-PuroR weresubjected to caspase-1 immunoprecipitation. Immunoprecipitated sampleswere then separated in a 12% SDS-PAGE and transferred into a PVDFmembrane. Membranes were probed with either anti-caspase-1 antibody oranti-T2A antibody (to detect TatCARD).

Expression of TatCARD in stable cell lines. The human monocytic cellline THP-1 was selected for its known ability to secrete IL-1β uponIFN-γ and LPS induction. Cells were transduced with eitherLV/EF1-MCS-T2A-PuroR or LV/EF1-TatCARD-T2A-PuroR and selected with 1microg/mL of puromycin for at least 2 week. A sample of each stable cellline and a sample of non-transduced cells (control) were lysed forprotein extraction. Protein lysate was run in a 12% SDS-PAGE assay andthe expression of the transgene was detected using western blot with ananti-T2A antibody. This antibody detects the attached fragment of the 2Apeptide that remains fused to the transgene after self-cleavage of theT2A peptide. The TatCARD was detected in the protein lysate ofTatCARD-THP1 cells only. Actin detection was used as a loading control.

FIG. 4. Expression of the fused protein TatCARD inhibits the LPS-inducedsecretion of IL-1β in THP-1 cells. THP-1 cells stably expressing eitherthe PuroR alone or the TatCARD fused protein were grown in the presenceor absence of IFN-γ for 4 hours followed by incubation with or withoutLPS for 18 hours. Conditioned media was harvested afterwards and thelevels of IL-1β were quantified by enzyme linked immunosorbent assay(ELISA). The levels of IL-1β in the absence of any stimulation (Media)were almost undetectectable by the assay. Cells expressing the TatCARDfusion protein showed a significant inhibition of IL-1β secretion whencompared to cell expressing only the PuroR gene (Vector).

FIG. 5. Expression of TatCARD inhibits the secretion of IL-1b in an invitro model of RPE inflammation. ARPE-19 cells expressing either thePuroR or TatCARD were stimulated with 4HNE (30 μM). The concentration ofIL-1β in their conditioned media was quantified by ELISA.

Expression of the fused protein TatCARD inhibits the 4-HNE-inducedsecretion of IL-1β in ARPE-19 cells. The human retinal pigmentedepithelium (RPE) like cells ARPE-19 were transduced with eitherLV/EF1-MCS-T2A-PuroR or LV/EF1-TatCARD-T2A-PuroR and selected for theirresistance against puromycin. Resistant cells were grown in the presenceor absence (Media) of 4-hydroxynonenal (an active aldehyde produced indry-AMD) for 24 hours. Afterwards the levels of IL-1β in the conditionedmedia were determined by ELISA as done in previous experiments. Theexpression of the TatCARD fused protein decreased the levels of 4-HNEinduced secretion of IL-1β when compared to vector control cells.

FIG. 6. Development of an AAV vector delivering a secretable form of theTatCARD fused protein. The fused protein TatCARD DNA sequence was clonedin frame and downstream of the IgK signal peptide and GFP gene in apuc57 plasmid. The new construct IgK signal peptide-GFP-FC-TatCARD wasthen cloned in an AAV plasmid. This plasmid contains the chicken-betaactin (smCBA) promoter flanked by the terminal repeats (TRs) sequencesof the AAV virus. The IgK signal peptide fused to the GFP allows thisgene to be targeted for secretion upon translation. This secreted GFP(sGFP) is, in-turn, linked to the TatCARD fused protein by a furincleavage (FC) site which allows the cleavage and separation of sGFP fromthe TatCARD upon cleavage at the cell membrane by the furin enzyme.Overall, this vector will deliver a secretable form of the fused TatCARDprotein.

FIG. 7a . The sGFP-TatCARD protein construct has a punctate pattern invitro. HEK293T cells were transfected with either pTR-smCBA-GFP orpTR-smCBA-sGFP-TatCARD. Cells were imaged 48 hours post-transfectionusing fluorescence microscopy to determine the distribution of GFP inboth cell groups. Cells transfected with the GFP plasmid showed atypical cytoplasmic distribution of GFP, whereas the cells transfectedwith the sGFP-TatCARD plasmid showed a distinct punctate patterncharacteristic of molecules that are actively being targeted for cellsecretion.

FIG. 7b . Detection of GFP in the media of cells transfected with thepTR-smCBA-sGFP-TatCARD plasmid. Transfected HEK293T cells were lysed at48 hours post-transfection after imaging, and their conditioned mediaharvested. Cells were lysed for protein extraction as done previously.Their corresponding conditioned media was concentrated sequentiallyusing a 50 KDa concentrator and collecting the eluted fraction. Thisfraction was then subjected to a second concentration step by using a 3KDa concentrator. The concentrated media containing molecules smallerthan 50 KDa but larger than 3 KDa was prepared for SDS-PAGE. Cell lysate(Cell, 30 microg. per lane) and concentrated media (Media, 15 microg.per lane) were separated in a 12% SDS-PAGE. The presence of GFP wasdetermined by western blot with an anti-GFP antibody. To bands of equalintensity were observed in the cell lysate of sGFP-TatCARD correspondingto the sGFP-TatCARD and the sGFP products. One band of approximately thesame size of the GFP was observed in the media of sGFP-TatCARD, thussuggesting the secretion and cleavage of the sGFP-TatCARD construct.Actin was used as a loading control.

FIG. 8. In vitro testing of the AAV1/smCBA-sGFP-TatCARD vector a HEK293Twere transduced with 10,000 vgc/cell of either AAV1/smCBA-GFP orAAV1/smCBA-sGFP-TatCARD. Growth media was replaced with 2 mL oflow-protein media 48 hrs after transduction. Conditioned media and cellswere harvested 72 hrs after transduction. Protein was extracted fromcell lysates as done in previous experiments. Conditioned media wasconcentrated using a 3 KDa column by centrifugation. Using a 12%SDS-PAGE, samples were separated using 30 microg. of each lysate (lys.)and 20 microg. of each conditioned media (media). The presence ofTatCARD fused protein was determined by western blot using an anti-ASCantibody that recognizes the CARD domain of the ASC protein. Two bandswere detected only in the media of cells transduced withAAV1/smCBA-sGFP-TatCARD corresponding to the fused sGFP-TatCARD (˜37KDa) and the cleaved TatCARD products (˜15 KDa). Tubulin detection wasused as a loading control for cell lysate samples b. Amido blackstaining of PVDF membrane showing total protein loaded in each lane ofthe gel.

FIG. 9. The biological activity of TatCARD can be transferred in vitro.Conditioned media from cells transfected with either GFP or TatCARDexpressing plasmid was overlayed on wild type ARPE-19 cells. These cellswere then stimulated with 4HNE and the concentration of secreted IL-1βin the media was measured by ELISA.

FIG. 10a . Experimental design for testing theAAV2-QUAD-T419V/smCBA-sGFP-TatCARD vector in the endotoxin-induced mousemodel. C57BL6J mice were injected intravitreally with 3×10⁹ vectorgenomes delivering either GFP or TatCARD AAV2. Twenty days later geneexpression was determined by fluorescent fundoscopy. Nine daysafterwards mice received 25 ng of LPS intravitreally. Eyes wereenucleated 24 hours later and fixed for histological analysis.Infiltrative cells were then quantified by two individuals.

FIG. 10b . Fundus of animals injected with AAV vector. Mice wereevaluated 21 days after AAV injection by fluorescent fundoscopy. Eyesexpressing GFP showed a expression of GFP in individual cells. Eyesinjected with the sGFP-TatCARD delivering AAV vector showed a diffusedGFP expression representative of secreted GFP. Non-injected eye was usedas a negative control.

FIG. 10c . Representative histological differences between eyes injectedwith GFP and eyes injected with sGFP-TatCARD. Histological sections ofeyes injected with AAV vector delivering either GFP or sGFP-TatCARD.Bright field pictures were taken at 5× and 10× (original magnification)to demonstrate the presence of infiltrative cells within the vitreousbody.

FIG. 10d . Expression of the TatCARD gene product decreases the numberof infiltrating cells in the EIU mouse model. Infiltrative cells withinthe vitreous body were quantified by two individuals. Average valueswere compared using the student t-test for paired observations. There isstatistically significant decrease in the number of infiltrative cellsin eyes injected with the AAV vector delivering the sGFP-TatCARD gene.

DETAILED DESCRIPTION Definitions

“Agent” as used herein pertains to a CARD polypeptide or a CARDnucleotide that encodes a CARD polypeptide. Unless otherwise indicated,an agent may include a delivery vehicle that comprises a CARD nucleotidefor expression of a CARD polypeptide.

“Biocompatible” refers to a material that is substantially non-toxic tocells in vitro, e.g., if its addition to cells in culture results inless than or equal to 20% cell death. A material is consideredbiocompatible with respect to a recipient if it is substantiallynontoxic to the recipient's cells in the quantities and at the locationused, and also does not elicit or cause a significant deleterious oruntoward effect on the recipient's body, e.g., an immunological orinflammatory reaction, unacceptable scar tissue formation, etc.

“Biodegradable” means that a material is capable of being broken downphysically and/or chemically within cells or within the body of asubject, e.g., by hydrolysis under physiological conditions, by naturalbiological processes such as the action of enzymes present within cellsor within the body, etc., to form smaller chemical species which can bemetabolized and, optionally, reused, and/or excreted or otherwisedisposed of. Preferably a biodegradable compound is biocompatible.

“Concurrent administration” as used herein with respect to two or moreagents, e.g., therapeutic agents, is administration performed usingdoses and time intervals such that the administered agents are presenttogether within the body, or at a site of action in the body such aswithin the eye) over a time interval in less than de minimis quantities,i.e., in quantities sufficient to have a detectable biological effect orresponse. The time interval can be minutes, hours, days, weeks, etc.Accordingly, the agents may, but need not be, administered together aspart of a single composition. In addition, the agents may, but need notbe, administered simultaneously (e.g., within less than 5 minutes, orwithin less than 1 minute) or within a short time of one another (e.g.,less than 1 hour, less than 30 minutes, less than 10 minutes,approximately 5 minutes apart). According to various embodiments of theinvention agents administered within such time intervals may beconsidered to be administered at substantially the same time. One ofordinary skill in the art will be able to readily determine appropriatedoses and time interval between administration of the agents so thatthey will each be present at more than de minimis levels within the bodyor, preferably, at effective concentrations within the body. Whenadministered concurrently, the effective concentration of each of theagents to elicit a particular biological response may be less than theeffective concentration of each agent when administered alone, therebyallowing a reduction in the dose of one or more of the agents relativeto the dose that would be needed if the agent was administered as asingle agent. The effects of multiple agents may, but need not be,additive or synergistic. The agents may be administered multiple times.

An “effective amount” of an active agent refers to the amount of theactive agent sufficient to elicit a desired biological response. As willbe appreciated by those of ordinary skill in this art, the absoluteamount of a particular agent that is effective may vary depending onsuch factors as the desired biological endpoint, the agent to bedelivered, the target tissue, etc. Those of ordinary skill in the artwill further understand that an “effective amount” may be administeredin a single dose, or may be achieved by administration of multipledoses. For example, for diseases or conditions involving the eye, aneffective amount may be an amount sufficient to achieve one or more ofthe following: (i) prevent drusen formation; (ii) cause a reduction indrusen number and/or size (drusen regression); (iii) cause a reductionin or prevent lipofuscin deposits; (iv) prevent visual loss or slow therate of visual loss; (v) prevent or slow the rate of choroidalneovascularization; (vi) cause a reduction in size and/or number oflesions characterized by choroidal neovascularization; (vii) improvevisual acuity and/or contrast sensitivity; (viii) prevent or reduce therate of photoreceptor or RPE cell atrophy or apoptosis; (ix) prevent orslow progression from the wet to the dry form of AMD.

“Local administration” or “local delivery”, in reference to delivery ofa composition, formulation, or device of the invention, refers todelivery that does not rely upon transport of the agent to its intendedtarget tissue via the vascular or lymphatic system from a site ofadministration that is remote from the intended target tissue. The agentis delivered directly to its intended target tissue or in the vicinitythereof, e.g. by injection or implantation. It will be appreciated thata small amount of the delivered agent may enter the vascular system andmay ultimately reach the target tissue via the vascular system.

“Macular degeneration related condition” refers to any of a number ofdisorders and conditions in which the macula degenerates or losesfunctional activity. The degeneration or loss of functional activity canarise as a result of, for example, cell death, decreased cellproliferation, loss of normal biological function, or a combination ofthe foregoing. Macular degeneration can lead to and/or manifest asalterations in the structural integrity of the cells and/orextracellular matrix of the macula, alteration in normal cellular and/orextracellular matrix architecture, and/or the loss of function ofmacular cells. The cells can be any cell type normally present in ornear the macula including RPE cells, photoreceptors, and capillaryendothelial cells. AMD is the major macular degeneration relatedcondition, but a number of others are known including, but not limitedto, Best macular dystrophy, Sorsby fundus dystrophy, MallatiaLeventinese and Doyne honeycomb retinal dystrophy.

“Ocular device” refers to a drug delivery device that has appropriatestructure, dimensions, shape, and/or configuration and is made ofappropriate materials so that it may be placed in or on the surface ofthe eye without causing unacceptable interference with the physiology orfunctioning of the eye. Preferably placement of an ocular device doesnot significantly disrupt vision. An ocular device is typically a solidor semi-solid article of manufacture and is typically macroscopic, i.e.,visible with the naked eye.

“Ocular neovascularization” (ONV) is used herein to refer to choroidalneovascularization or retinal neovascularization, or both.

“Polypeptide”, as used herein, refers to a polymer of amino acids and/oramino acid analogs which may or may not be modified. Various amino acidanalogs and modifications are described herein. A polypeptide may becyclic or linear and may be branched or unbranched. The term “amino acidsequence” or “polypeptide sequence” as used herein can refer to thepolypeptide material itself and is not restricted to the sequenceinformation (i.e. the succession of letters or three letter codes chosenamong the letters and codes used as abbreviations for amino acid names)that biochemically characterizes a polypeptide. For purposes of thedisclosure the use of the term “polypeptide” and “protein” areinterchangeable unless specifically noted otherwise.

“Purified”, as used herein, means separated from many other compounds orentities. A compound or entity may be partially purified, substantiallypurified, or pure. A compound or entity is considered pure when it isremoved from substantially all other compounds or entities, i.e., ispreferably at least about 90%, more preferably at least about 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure. A partiallyor substantially purified compound or entity may be removed from atleast 50%, at least 60%, at least 70%, or at least 80% of the materialwith which it is naturally found, e.g., cellular material such ascellular proteins and/or nucleic acids.

“Retinal neovascularization” (RNV) refers to the abnormal development,proliferation, and/or growth of retinal blood vessels, e.g., on theretinal surface.

“Sequential administration” of two or more agents refers toadministration of two or more agents to a subject such that the agentsare not present together in the subject's body at greater than deminimis concentrations. Administration of the agents may, but need not,alternate. Each agent may be administered multiple times.

“Subject”, as used herein, refers to an individual to whom an agent isto be delivered, e.g., for experimental, diagnostic, and/or therapeuticpurposes. Preferred subjects are mammals, particularly domesticatedmammals (e.g., dogs, cats, etc.), primates, or humans.

“Significant sequence homology” as applied to an amino acid sequencemeans that the sequence displays at least approximately 20% identical orconservatively replaced amino acids, preferably at least approximately30%, at least approximately 40%, at least approximately 50%, at leastapproximately 60% identical or conservatively replaced amino acids,desirably at least approximately 70% identical or conservativelyreplaced amino acids, more desirably at least approximately 80%identical or conservatively replaced amino acids, and most desirably atleast approximately 90% amino acid identical or conservatively replacedamino acids relative to a reference sequence. When two or more sequencesare compared, any of them may be considered the reference sequence. %identity can be calculated using a FASTA or BLASTP algorithm, usingdefault parameters. A PAM250 or BLOSUM62 matrix may be used. Forpurposes of calculating % identical or conservatively replaced residues,a conservatively replaced residue is considered identical to the residueit replaces. Conservative replacements may be defined in accordance withStryer, L, Biochemistry, 3rd ed., 1988, according to which amino acidsin the following groups possess similar features with respect to sidechain properties such as charge, hydrophobicity, aromaticity, etc. (1)Aliphatic side chains: G, A, V, L, I; (2) Aromatic side chains: F, Y, W;(3) Sulfur-containing side chains: C, M; (4) Aliphatic hydroxyl sidechains: S, T; (5) Basic side chains: K, R, H; (6) Acidic amino acids: D,E, N, Q; (7) Cyclic aliphatic side chain: P

“Substantial sequence homology” as applied to a sequence means that thesequence displays at least approximately 60% identity, desirably atleast approximately 70% identity, more desirably at least approximately80% identity, and most desirably at least approximately 90% identityrelative to a reference sequence. When two or more sequences arecompared, any of them may be considered the reference sequence. %identity can be calculated using a FASTA, BLASTN, or BLASTP algorithm,depending on whether amino acid or nucleotide sequences are beingcompared. Default parameters may be used. A PAM250 or BLOSUM62 matrixmay be used.

A “sustained release formulation” is a composition of matter thatcomprises a therapeutic agent as one of its components and furthercomprises one or more additional components, elements, or structureseffective to provide sustained release of the therapeutic agent,optionally in part as a consequence of the physical structure of theformulation. Sustained release is release or delivery that occurs eithercontinuously or intermittently over a period of time e.g., at least 1,2, 4, or 6 weeks, at least 1, 2, 3, 4, 6, 8, 10, 12, 15, 18, or 24months, or longer.

“Treating” or “treatment of” as used herein, refers to providing anytype of medical or surgical management to a subject. Treating caninclude, but is not limited to, administering a composition comprising atherapeutic agent to a subject. “Treating” includes any administrationor application of a agent or composition of the invention to a subjectfor purposes such as curing, reversing, alleviating, reducing theseverity of, inhibiting the progression of, or reducing the likelihoodof a disease, disorder, or condition or one or more symptoms ormanifestations of a disease, disorder or condition. In a specificexample, a composition of this invention can be administered to asubject who has developed a macular degeneration related condition riskof developing an infection relative to a member of the generalpopulation. A composition of this invention can be administered to asubject who has developed an eye disorder such as exudative ornon-exudative AMD or diabetic retinopathy or is at increased risk or whohas exhibited symptoms of developing such a disorder relative to amember of the general population. A composition of this invention can beadministered prophylactically, i.e., before development of any symptomor manifestation of the condition. Typically in this case the subjectwill be at risk of developing the condition. Treating also may comprisetreating a subject exhibiting symptoms of a certain disease orcondition.

A variety of factors including oxidative stress, inflammation with apossible autoimmune component, genetic background (e.g., mutations), andenvironmental or behavioral features such as smoking and diet maycontribute to the pathogenesis of AMD in manners that are as yet poorlyunderstood (Zarbin, M A, Arch Opthalmol. 122:598-614, 2004). Regardlessof the underlying etiology, the clinical hallmark of AMD is theappearance of drusen, localized deposits of lipoproteinaceous materialthat accumulate in the space between the RPE and Bruch's membrane, whichseparates the RPE from the choroidal vessels (choriocapillaris). Drusenare typically the earliest clinical finding in AMD. The existence ofmacular drusen is a strong risk factor for the development of both wetand dry forms of AMD (Ambati, J., et al., Surv. Opthalmol., 48(3):257-293, 2003).

Ocular inflammation can affect a large number of eye structuresincluding the conjunctiva, cornea, episclera, sclera, uveal tract,retina, vasculature, optic nerve, and orbit. Evidence of ocularinflammation can include the presence of inflammation-associated cellssuch as white blood cells (e.g., neutrophils, macrophages) in the eye,the presence of endogenous inflammatory mediators known in the art, oneor more symptoms such as eye pain, redness, light sensitivity, blurredvision and floaters, etc. Uveitis is a general term that refers toinflammation in the uvea of the eye, e.g., in any of the structures ofthe uvea, including the iris, ciliary body or choroid. Specific types ofuveitis include iritis, iridocyclitis, cyclitis, pars planitis andchoroiditis. Uveitis can arise from a number of different causes and isassociated with a number of different diseases, including, but notlimited to, rheumatic diseases such as rheumatic diseases (e.g.,ankylosing spondylitis and juvenile rheumatoid arthritis), certaininfectious diseases such as tuberculosis and syphilis, other conditionssuch as sarcoidosis, systemic lupus erythematosus, chemical injury,trauma, surgery, etc. Keratis refers to inflammation of the cornea.Keratitis has a diverse array of causes including bacterial, viral, orfungal infection, trauma, and allergic reaction. Amoebic infection ofthe cornea, e.g., caused by Acanthamoeba, is a particular problem forcontact lens wearers. Scleritis refers to inflammation of the sclera.Uveitis, keratitis, and scleritis, and methods for their diagnosis arewell known in the art. Symptoms of the various inflammatory conditionsthat affect the eye can include, but are not limited to, eye pain,redness, light sensitivity, tearing, blurred vision, floaters. Ocularinflammation of various types is well known to occur in association witha variety of local or systemic diseases, some of which are noted above.In some instances the cause may remain unknown.

DETAILED DESCRIPTION

According to one embodiment, provided is a method for the prevention,amelioration, or treatment of a disease or condition associated withoxidative stress or inflammation in a subject comprising administrationof a therapeutically effective amount of an agent to the subject,wherein the agent is a CARD protein. In a specific embodiment, the CARDprotein is a TatCARD protein, or a polypeptide having substantialsequence homology therewith.

In a specific embodiment, provided is a method of preventing,ameliorating or treating retinal and RPE inflammation that involvesadministration of a CARD protein. In a more specific embodiment, theCARD protein is secreted from cells transfected with an AAV vectorengineered to express CARD, and in particular TatCARD.

In another embodiment, provided is a viral vector engineered to expressTatCARD. In specific embodiments, the viral vector is an AAV plasmid ora lentiviral plasmid.

In a further embodiment, provided are cells stably transfected with anucleotide sequence encoding a CARD protein. In a specific embodiment,the CARD protein is TatCARD.

In a particular embodiment, the invention pertains to a method oftreating a disease associated with inflammation or oxidative stressinvolving the eye. Specific examples of such diseases include but arenot limited to macular degeneration, age-related macular degeneration(AMD), geographic atrophy, wet AMD, dry AMD, drusen formation, dry eye,diabetic retinopathy, vitreoretinopathy, corneal inflammation, uveitis,ocular hypertension or glaucoma.

Vectors

In some embodiments, viral vectors are used to transfect cells with aCARD expression construct. In a particular embodiment, adeno-associatedviral vectors are used. Other vectors of the invention used in vitro, invivo, and ex vivo include viral vectors, such as retroviruses (includinglentiviruses), herpes viruses, alphavirus, adenovirus, vaccinia virus,papillomavirus, or Epstein Barr virus (EBV).

Methods for constructing and using viral vectors are known in the art(see, e.g., Miller and Rosman, BioTechniques 1992, 7:980-990). Inaccordance with the present invention there may be employed conventionalmolecular biology, microbiology, and recombinant DNA techniques withinthe skill of the art. Such techniques are well-known and are explainedfully in the literature. See, e.g., Sambrook, Fritsch and Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Various companies produce viral vectors commercially, including but byno means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors), CellGenesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors) and Origene(Rockville, Md.).

In certain embodiments, the viral vectors of the invention arereplication defective, that is, they are unable to replicateautonomously in the target cell. Preferably, the replication defectivevirus is a minimal virus, i.e., it retains only the sequences of itsgenome which are necessary for target cell recognition and encapsidatingthe viral genome. Replication defective virus is not infective afterintroduction into a cell. Use of replication defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Thus, a specifictissue can be specifically targeted. Examples of particular vectorsinclude, but are not limited to, defective herpes virus vectors (see,e.g., Kaplitt et al., Molec. Cell. Neurosci. 1991, 2:320-330; PatentPublication RD 371005 A; PCT Publications No. WO 94/21807 and WO92/05263), defective adenovirus vectors (see, e.g.,Stratford-Perricaudet et al., J. Clin. Invest. 1992, 90:626-630; LaSalle et al., Science 1993, 259:988-990; PCT Publications No. WO94/26914, WO 95/02697, WO 94/28938, WO 94/28152, WO 94/12649, WO95/02697, and WO 96/22378), and defective adeno-associated virus vectors(Samulski et al., J. Virol. 1987, 61:3096-3101; Samulski et al., J.Virol. 1989, 63:3822-3828; Lebkowski et al., Mol. Cell. Biol. 1988,8:3988-3996; PCT Publications No. WO 91/18088 and WO 93/09239; U.S. Pat.Nos. 4,797,368 and 5,139,941; European Publication No. EP 488 528).

Adeno-associated virus-based vectors. The adeno-associated viruses (AAV)are DNA viruses of relatively small size which can integrate, in astable and site-specific manner, into the genome of the cells which theyinfect. They are able to infect a wide spectrum of cells withoutinducing any effects on cellular growth, morphology or differentiation,and they do not appear to be involved in human pathologies. The AAVgenome has been cloned, sequenced and characterized. The use of vectorsderived from the AAVs for transferring genes in vitro and in vivo hasbeen described (see PCT Publications No. WO 91/18088 and WO 93/09239;U.S. Pat. Nos. 4,797,368 and 5,139,941; EP Publication No. 488 528). Thereplication defective recombinant AAVs according to the invention can beprepared by cotransfecting a plasmid containing the nucleic acidsequence of interest flanked by two AAV inverted terminal repeat (ITR)regions, and a plasmid carrying the AAV encapsidation genes (rep and capgenes), into a cell line which is infected with a human helper virus(e.g., an adenovirus). The AAV recombinants which are produced are thenpurified by standard techniques.

Adenovirus-based vectors. Adenoviruses are eukaryotic DNA viruses thatcan be modified to efficiently deliver a nucleic acid of the inventionto a variety of cell types. Various serotypes of adenovirus exist. Ofthese serotypes, preference is given, within the scope of the presentinvention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5)or adenoviruses of animal origin (see PCT Publication No. WO94/26914).Those adenoviruses of animal origin which can be used within the scopeof the present invention include adenoviruses of canine, bovine, murine(e.g., Mav1 [Beard et al., Virology, 1990, 75:81]), ovine, porcine,avian, and simian (e.g., SAV) origin. Preferably, the adenovirus ofanimal origin is a canine adenovirus, more preferably a CAV2 adenovirus(e.g., Manhattan or A26/61 strain [ATCC Accession No. VR-800]). Variousreplication defective adenovirus and minimum adenovirus vectors havebeen described (PCT Publications No. WO94/26914, WO95/02697, WO94/28938,WO94/28152, WO94/12649, WO95/02697, WO96/22378). The replicationdefective recombinant adenoviruses according to the invention can beprepared by any technique known to the person skilled in the art(Levrero et al., Gene, 1991, 101:195; EP Publication No. 185 573;Graham, EMBO J., 1984, 3:2917; Graham et al., J. Gen. Virol., 1977,36:59). Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques, which are well known to one ofordinary skill in the art.

Retroviral vectors. In another embodiment, the invention providesretroviral vectors, e.g., as described in Mann et al., Cell 1983,33:153; U.S. Pat. Nos. 4,650,764, 4,980,289, 5,124,263, and 5,399,346;Markowitz et al., J. Virol. 1988, 62:1120; EP Publications No. 453 242and 178 220; Bernstein et al. Genet. Eng. 1985, 7:235; McCormick,BioTechnology 1985, 3:689; and Kuo et al., 1993, Blood, 82:845. Theretroviruses are integrating viruses which infect dividing cells. Theretrovirus genome includes two LTRs, an encapsidation sequence and threecoding regions (gag, pol and env). Replication defective non-infectiousretroviral vectors are manipulated to destroy the viral packagingsignal, but retain the structural genes required to package theco-introduced virus engineered to contain the heterologous gene and thepackaging signals. Thus, in recombinant replication defective retroviralvectors, the gag, pol and env genes are generally deleted, in whole orin part, and replaced with a heterologous nucleic acid sequence ofinterest. These vectors can be constructed from different types ofretroviruses, such as HIV (human immuno-deficiency virus), MoMuLV(murine Moloney leukaemia virus), MSV (murine Moloney sarcoma virus),HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Roussarcoma virus), and Friend virus. Suitable packaging cell lines havebeen described in the prior art, in particular, the cell line PA317(U.S. Pat. No. 4,861,719); the PsiCRIP cell line (PCT Publication No. WO90/02806) and the GP+envAm-12 cell line (PCT Publication No. WO89/07150). In addition, recombinant retroviral vectors can containmodifications within the LTRs for suppressing transcriptional activityas well as extensive encapsidation sequences which may include a part ofthe gag gene (Bender et al., J. Virol. 1987, 61:1639). Recombinantretroviral vectors are purified by standard techniques known to thosehaving ordinary skill in the art.

Retrovirus vectors can also be introduced by DNA viruses, which permitsone cycle of retroviral replication and amplifies transfectionefficiency (see PCT Publications No. WO 95/22617, WO 95/26411, WO96/39036, WO 97/19182).

In a specific embodiment of the invention, lentiviral vectors can beused as agents for the direct delivery and sustained expression of atransgene in several tissue types, including brain, retina, muscle,liver, and blood. This subtype of retroviral vectors can efficientlytransduce dividing and nondividing cells in these tissues, and maintainlong-term expression of the gene of interest (for a review, see,Naldini, Curr. Opin. Biotechnol. 1998, 9:457-63; Zufferey, et al., J.Virol. 1998, 72:9873-80). Lentiviral packaging cell lines are availableand known generally in the art (see, e.g., Kafri, et al., J. Virol.,1999, 73: 576-584).

Non-viral vectors. In another embodiment, the invention providesnon-viral vectors that can be introduced in vivo, provided that thesevectors contain a targeting peptide, protein, antibody, etc. thatspecifically binds HALR. For example, compositions of synthetic cationiclipids, which can be used to prepare liposomes for in vivo transfectionof a vector carrying an anti-tumor therapeutic gene, are described inFeigner et. al., Proc. Natl. Acad. Sci. USA 1987, 84:7413-7417; Feignerand Ringold, Science 1989, 337:387-388; Mackey, et al., Proc. Natl.Acad. Sci. USA 1988, 85:8027-8031; and Ulmer et al, Science 1993,259:1745-1748. Useful lipid compounds and compositions for transfer ofnucleic acids are described, e.g., in PCT Publications No. WO 95/18863and WO96/17823, and in U.S. Pat. No. 5,459,127. Targeting peptides,e.g., laminin or HALR-binding laminin peptides, and proteins such asanti-HALR antibodies, or non-peptide molecules can be coupled toliposomes covalently (e.g., by conjugation of the peptide to aphospholipid or cholesterol; see also Mackey et al., supra) ornon-covalently (e.g., by insertion via a membrane binding domain ormoiety into the bilayer membrane).

Alphaviruses are well known in the art, and include without limitationEquine Encephalitis viruses, Semliki Forest virus and related species,Sindbis virus, and recombinant or ungrouped species (see Strauss andStrauss, Microbiol. Rev. 1994, 58:491-562, Table 1, p. 493).

As used herein the term “replication deficient virus” has its ordinarymeaning, i.e., a virus that is propagation incompetent as a result ofmodifications to its genome. Thus, once such recombinant virus infects acell, the only course it can follow is to express any viral andheterologous protein contained in its genome. In a specific embodiment,the replication defective vectors of the invention may contain genesencoding nonstructural proteins, and are self-sufficient for RNAtranscription and gene expression. However, these vectors lack genesencoding structural proteins, so that a helper genome is needed to allowthem to be packaged into infectious particles. In addition to providingtherapeutically safe vectors, the removal of the structural proteinsincreases the capacity of these vectors to incorporate more than 6 kb ofheterologous sequences. In another embodiment, propagation incompetenceof the adenovirus vectors of the invention is achieved indirectly, e.g.,by removing the packaging signal which allows the structural proteins tobe packaged in virions being released from the packaging cell line. Asdiscussed above, viral vectors used to transfect cells and express CARDpolypeptide may be used, and in a specific embodiment, the viral vectorsinvolve a replication deficient virus.

Other Delivery Vehicles

Many nonviral techniques for the delivery of a nucleic acid sequenceinto a cell can be used, including direct naked DNA uptake (e.g., Wolffet al., Science 247: 1465-1468, 1990), receptor-mediated DNA uptake,e.g., using DNA coupled to asialoorosomucoid which is taken up by theasialoglycoprotein receptor in the liver (Wu and Wu, J. Biol. Chem. 262:4429-4432, 1987; Wu et al., J. Biol. Chem. 266: 14338-14342, 1991), andliposome-mediated delivery (e.g., Kaneda et al., Expt. Cell Res. 173:56-69, 1987; Kaneda et al., Science 243: 375-378, 1989; Zhu et al.,Science 261: 209-211, 1993). Many of these physical methods can becombined with one another and with viral techniques; enhancement ofreceptor-mediated DNA uptake can be effected, for example, by combiningits use with adenovirus (Curiel et al., Proc. Natl. Acad. Sci. USA 88:8850-8854, 1991; Cristiana et al., Proc. Natl. Acad. Sci. USA 90:2122-2126, 1993). Other examples include stem cells such as mesenchymalstem cells, hematopoietic stem cells, cardiac stem cells or neural stemcells, embryonic stem cells that have been engineered to express asequence of interest.

Dosing, Delivery and Formulations

Further expounding on the definitions provided above, informationregarding dosages and dosing is provided here.

Suitable dosage amounts and dosing regimens can be determined by theattending physician and may depend on the severity of the condition aswell as the general age, health and weight of the patient to be treated.

Dosing may occur at intervals of minutes, hours, days, weeks, months oryears or continuously over any one of these periods. Suitable dosagesmay lie within the range of about 0.1 ng per kg of body weight to 1 gper kg of body weight per dosage, such as is in the range of 1 mg to 1 gper kg of body weight per dosage. In one embodiment, the dosage may bein the range of 1 mg to 500 mg per kg of body weight per dosage. Inanother embodiment, the dosage may be in the range of 1 mg to 250 mg perkg of body weight per dosage. In yet another embodiment, the dosage maybe in the range of 1 mg to 100 mg per kg of body weight per dosage, suchas up to 50 mg per body weight per dosage.

The agent of the invention may be administered in a single dose or aseries of doses. While it is possible for the active ingredient to beadministered alone, it is preferable to present it as a composition,preferably as a pharmaceutical composition. The formulation of suchcompositions is well known to those skilled in the art. The compositionmay contain any suitable carriers, diluents or excipients. These includeall conventional solvents, dispersion media, fillers, solid carriers,coatings, antifungal and antibacterial agents, dermal penetrationagents, surfactants, isotonic and absorption agents and the like. Itwill be understood that the compositions of the invention may alsoinclude other supplementary physiologically active agents.

The carrier must be pharmaceutically “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notinjurious to the patient. The compositions may conveniently be presentedin unit dosage form and may be prepared by any methods well known in theart of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general, the compositions are preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product.

The methods of treatment described herein may involve administration ofcompositions comprising delivery vehicles, such as expression vectors,to the eye. Methods of treatment may also involve formulating CARDpolypeptides into compositions for application to the eye of patients inneed of therapy. Thus, such compositions are adapted for pharmaceuticaluse as an injectable agent, or as an eye drop or in contact lenses,inserts or the like, as described in greater detail below. Accordingly,formulation of compound into sterile water containing any desireddiluents, salts, pH modifying materials and the like as are known topersons skilled in the pharmaceutical formulations art may be performedin order to achieve a solution compatible with administration to theeye. It may be that injectables, eye drops, inserts, contact lenses,gels and other liquid forms may require somewhat different formulations.All such formulations consistent with direct administration to the eyeare comprehended hereby.

The compositions may also have antioxidants in ranges that varydepending on the kind of antioxidant used. The usage also depends on theamount of antioxidant needed to allow at least 2 years shelf-life forthe pharmaceutical composition. One or more antioxidants may be includedin the formulation. Certain commonly used antioxidants have maximumlevels allowed by regulatory authorities. As such, the amount ofantioxidant(s) to be administered should be enough to be effective whilenot causing any untoward effect. Such doses may be adjusted by aphysician as needed, within the maximum levels set by regulatoryauthorities, and is well within the purview of the skilled artisan todetermine the proper and effective dose. Reasonable ranges are about0.01% to about 0.15% weight by volume of EDTA, about 0.01% to about 2.0%weight volume of sodium sulfite, and about 0.01% to about 2.0% weight byvolume of sodium metabisulfite. One skilled in the art may use aconcentration of about 0.1% weight by volume for each of the above.N-Acetylcysteine may be present in a range of about 0.1% to about 5.0%weight by volume, with about 1% to about 10% of hydroxylamineconcentration being preferred. Ascorbic acid or salt may also be presentin a range of about 0.1% to about 5.0% weight by volume with about 1% toabout 10% weight by volume of hydroxylamine concentration preferred.Other sulfhydryls, if included, may be the same range as forN-acetylcysteine. Other exemplary compounds include mercaptopropionylglycine, N-acetyl cysteine, β-mercaptoethylamine, glutathione andsimilar species, although other anti-oxidant agents suitable for ocularadministration, e.g., ascorbic acid and its salts or sulfite or sodiummetabisulfite may also be employed.

A buffering agent may be used to maintain the pH of eye dropformulations in the range of about 4.0 to about 8.0; so as to minimizeirritation of the eye. In certain embodiments, the pH is maintained atabout 3.5 to about 6.0, preferably about 4.0 to about 5.5, in order toensure that most of the hydroxylamine is in its protonated form forhighest aqueous solubility. The buffer may be any weak acid and itsconjugate base with a pKa of about 4.0 to about 5.5; e.g., aceticacid/sodium acetate; citric acid/sodium citrate. The pKa of thehydroxylamines is about 6.0. For direct intravitreal or intraocularinjection, formulations should be at pH 7.2 to 7.5, preferably at pH7.3-7.4.

The ophthalmologic compositions may also include tonicity agentssuitable for administration to the eye. Among those suitable is sodiumchloride to make formulations of the present invention approximatelyisotonic with 0.9% saline solution.

In certain embodiments, the compositions are formulated with viscosityenhancing agents. Exemplary agents are hydroxyethylcellulose,hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone. Theviscosity agents may be present in the compounds up to about 2.0% weightby volume. It may be preferred that the agents are present in a rangefrom about 0.2% to about 0.5% weight by volume. A preferred range forpolyvinylpyrrolidone may be from about 0.1% to about 2.0% weight byvolume. One skilled in the art may prefer any range established asacceptable by the Food and Drug Administration.

The compounds used in accordance with the methods of the invention mayhave co-solvents added if needed. Suitable cosolvents may includeglycerin, polyethylene glycol (PEG), polysorbate, propylene glycol,mannitol and polyvinyl alcohol. The presence of the co-solvents mayexist in a range of about 0.2% to about 4.0% weight by volume. It may bepreferred that mannitol may be formulated in the compounds of theinvention in a range of about 0.5% to about 4.0% weight by volume. Itmay also be preferred that polyvinyl alcohol may be formulated in thecompounds of the invention in a range of about 0.1% to about 4.0% weightby volume. One skilled in the art may prefer ranges established asacceptable by the Food and Drug Administration.

Preservatives may be used in the invention within particular ranges.Among those preferred are up to 0.013% weight by volume of benzalkoniumchloride, up to 0.013% weight by volume of benzethonium chloride, up to0.5% weight by volume of chlorobutanol, up to 0.004% weight by volume orphenylmercuric acetate or nitrate, up to 0.01% weight by volume ofthimerosal, and from about 0.01% to about 0.2% weight by volume ofmethyl or propylparabens.

Formulations for injection are preferably designed for single-useadministration and do not contain preservatives. Injectable solutionsshould have isotonicity equivalent to 0.9% sodium chloride solution(osmolality of 290-300 mOsmoles). This may be attained by addition ofsodium chloride or other co-solvents as listed above, or excipients suchas buffering agents and antioxidants, as listed above. Injectableformulations are sterilized and, in one embodiment, supplied insingle-use vials or ampules. In another embodiment, injectable productsmay be supplied as sterile, freeze-dried solids for reconstitution andsubsequent injection.

The tissues of the anterior chamber of the eye are bathed by the aqueoushumor, while the retina is under continuous exposure to the vitreous.These fluids/gels exist in a highly reducing redox state because theycontains antioxidant compounds and enzymes. Therefore, it may beadvantageous to include a reducing agent in the ophthalmologiccompositions formulated in accordance with the invention, or to doseseparately with a reducing agent to maintain the hydroxylamine in itsreduced form.

Preferred reducing agents may be N-acetylcysteine, ascorbic acid or asalt form, and sodium sulfite or metabisulfite, with ascorbic acidand/or N-acetylcysteine or glutathione being particularly suitable forinjectable solutions. A combination of N-acetylcysteine and sodiumascorbate may be used in various formulations. A metal chelatorantioxidant, such as EDTA (ethylenediaminetetraacetic acid) or possiblyDTPA (diethylenetriaminepentaacetic acid) may also be added to keep thehydroxylamine in the reduced form in the eye drop formulation.

Compositions utilized in accordance with the methods of the inventionmay be delivered to the eye of a patient in one or more of severaldelivery modes known in the art. In a preferred embodiment, thecompositions are topically delivered to the eye in eye drops or washes.In another embodiment, the compositions are delivered in a topicalophthalmic ointment, which is particularly useful for treatingconditions of the cornea, conjuctiva or surrounding skin, such asdry-eye and blepharitis. In another embodiment, the compositions may bedelivered to various locations within the eye via periodicsubconjunctival or intraocular injection, or by infusion in anirrigating solution such as BSS® or BSS PLUS® (Alcon USA, Fort Worth,Tex.) or by using pre-formulated solutions of the viral vectors (orother delivery expression vectors) or CARD protein (e.g. TatCARD) inexcipients such as BSS® or BSS PLUS®. In one embodiment, the use of thecompounds of the invention in vitrectomy may be effective in reducing orpreventing the development of vitrectomy-associated cataracts.

Alternatively, the compositions may be applied in other ophthalmologicdosage forms known to those skilled in the art, such as pre-formed or insitu-formed gels or liposomes, for example as disclosed in U.S. Pat. No.5,718,922 to Herrero-Vanrell. A direct injection of drugs into thevitreous body used for treating diseases has been used, in whichmicrospheres or liposomes were used to release drugs slowly (Moritera,T. et al. “Microspheres of biodegradable polymers as a drug-deliverysystem in the vitreous” Invest. Ophthalmol. Vis. Sci. 199132(6):1785-90).

In another embodiment, the composition may be delivered to or throughthe lens of an eye in need of treatment via a contact lens (e.g.,Lidofilcon B, Bausch & Lomb CW79 or DELTACON (Deltafilcon A) or otherobject temporarily resident upon the surface of the eye. For example,U.S. Pat. No. 6,410,045 describes a contact lens-type drug deliverydevice comprising a polymeric hydrogel contact lens containing drugsubstance in a concentration of between 0.05% and 0.25% by weightabsorbed in said contact lens which is capable of being delivered intothe ocular fluid.

In other embodiments, supports such as a collagen corneal shield (e.g.,B10-COR dissolvable corneal shields, Summit Technology, Watertown,Mass.) can be employed. The compositions can also be administered byinfusion into the eyeball, either through a cannula from an osmotic pump(ALZET®, Alza Corp., Palo Alto, Calif.) or by implantation oftimed-release capsules (OCCUSENT®) or biodegradable disks (OCULEX®,OCUSERT®) which contain the compositions. These routes of administrationhave the advantage of providing a continuous supply of the compositionto the eye. This may be an advantage for local delivery of thehydroxylamine compounds to the cornea and aqueous humor, for example.

Several other types of ocular devices/delivery systems are availablethat are particularly suitable for delivering pharmaceuticalcompositions to the interior or posterior of the eye. For instance, U.S.Pat. No. 6,154,671 to Parel et al. discloses a device for transferring amedicament into the eyeball by iontophoresis. The device utilizes areservoir for holding the active agent, which contains at least oneactive surface electrode facing the eye tissue lying at the periphery ofthe cornea. The reservoir also has a return electrode in contact withthe patient's partly closed eyelids. U.S. Pat. No. 5,869,079 to Wong etal. discloses combinations of hydrophilic and hydrophobic entities in abiodegradable sustained release ocular implant. In addition, U.S. Pat.No. 6,375,972 to Guo et al., U.S. Pat. No. 5,902,598 to Chen et al.,U.S. Pat. No. 6,331,313 to Wong et al., U.S. Pat. No. 5,707,643 to Oguraet al., U.S. Pat. No. 5,466,233 to Weiner et al. and U.S. Pat. No.6,251,090 to Avery et al. each describes intraocular implant devices andsystems that may be used to deliver pharmaceutical compositionscomprising compounds of the present invention.

Other examples of ocular devices pertain to ocular implants for drugdelivery are known in the art. Such devices could be loaded with agentsof the invention for delivery to the eye. The following is anon-limiting list of representative examples ocular devices foradministration to the eye:

-   -   U.S. Pat. No. 6,726,918 describes methods for treating        inflammation-mediated conditions of the eye comprising:        implanting into the vitreous of the eye of an individual a        biodegradable implant comprising a steroidal anti-inflammatory        agent and a biodegradable polymer, wherein the implant delivers        the agent to the vitreous in an amount sufficient to reach a        concentration equivalent to at least about 0.05 μg/ml        dexamethasone within about 48 hours and maintains a        concentration equivalent to at least about 0.03 μg/ml        dexamethasone for at least about three weeks.    -   U.S. Pat. No. 6,713,081 describes ocular implant devices for the        delivery of a therapeutic agent to an eye in a controlled and        sustained manner. Dual mode and single mode drug delivery        devices are illustrated and described. Implants suitable for        subconjunctival and intravitreal placement are described. The        patent also describes fabrication and implementation techniques        associated with the ocular implant devices.    -   U.S. Pat. No. 6,251,090 describes an intravitreal medicine        delivery device, method and implant device through which a wide        variety of beneficial medicines including drugs or other        pharmacological agents can be introduced into the vitreous        cavity over an extended period of time with only a single        initial surgery to implant the device. The device and method        minimize the surgical incision needed for implantation and avoid        future or repeated invasive surgery or procedures. Additional        amounts of the initial medicine can readily be introduced or the        medication can be varied or changed, as required. Furthermore,        the device and method allow the dosage delivered to the vitreous        cavity to be controlled and allows the patient to control the        timing of the delivery. The device is constructed so as to        filter medicines delivered to the cavity and also avoids damage        to or interference with other parts of the eye during        implantation or during use.    -   U.S. Pat. No. 5,824,072 describes biocompatible ocular implants        comprising active agents that are employed for introduction into        a suprachoroidal space or an avascular region of an eye for        therapeutic purposes. The administration of drugs is controlled        and maintained for long periods of time, while ensuring the        substantial absence of significant levels outside the site of        administration.    -   U.S. Pat. No. 5,773,019 describes a continuous release drug        delivery implant which, among other mentioned places, can be        mounted either on the outer surface of the eye or within the        eye. A drug core is covered by a polymer coating layer that is        permeable to the low solubility agent without being release rate        limiting.    -   U.S. Pat. No. 5,773,021 describes bioadhesive ophthalmic inserts        that are placed in the conjunctival sac. The inserts are        prepared by extrusion, thermoforming, or heat compression of a        polymeric material matrix and the drug to be delivered. The        polymeric matrix comprises a water-soluble biocompatible        polymer, such as hydroxyalkyl celluloses, maltodextrins,        chitosans, modified starches or polyvinyl alcohols; a        water-insoluble biocompatible polymer such as an alkyl        cellulose. Where applicable, a bioadhesive polymer such as        polyvinyl carboxylic acid type polymers or certain bioadhesive        polysaccharides or derivatives thereof may be used. The        ophthalmic inserts are characterized therein as intended for the        prolonged and controlled release of a medicinal substance.    -   U.S. Pat. Nos. 5,443,505 and 5,766,242 disclose implants        comprising active agents for introduction into a suprachoroidal        space or an avascular region of the eye, and describe placing        microcapsules and plaques comprising hydrocortisone into the        pars plana.    -   U.S. Pat. No. 5,378,475 describes a sustained-release implant        for insertion into the vitreous of the eye. The implant has a        first impermeable coating, such as ethylene vinyl acetate,        surrounding most, but not all, of a drug reservoir and a second        permeable coating, such as a permeable crosslinked polyvinyl        alcohol, disposed over the first coating including the region        where the first coating does not cover the drug reservoir, to        provide a location through which the drug can diffuse out of the        implant.    -   U.S. Pat. No. 5,725,493 describes an ocular implant device for        providing drugs to the vitreous cavity over a period of time.        The drug reservoir is attached to the outside of the eye with a        passageway permitting medicament to enter the vitreous cavity of        the eye.    -   U.S. Pat. No. 5,164,188 discloses encapsulated agents for        introduction into the suprachoroid of the eye, and describes        placing microcapsules and plaques comprising hydrocortisone into        the pars plana.    -   U.S. Pat. No. 4,997,652 discloses biodegradable ocular implants        comprising microencapsulated drugs, and describes implanting        microcapsules comprising hydrocortisone succinate into the        posterior segment of the eye.    -   U.S. Pat. No. 4,014,335 describes an ocular drug delivery device        placed in the cul-de-sac between the sclera and lower eyelid for        administering the drug and acting as a reservoir. The ocular        device is characterized therein as administering drug to the eye        in a controlled, continuous dosage rate over a prolonged time.        To accomplish this, the ocular device comprises a three-layered        laminate of polymeric materials holding the drug in a central        reservoir region of the laminate. The drug diffuses from the        reservoir through at least one of the polymeric layers of the        laminate.    -   U.S. Pat. No. 4,300,557 teaches a capsule which can be filled        with a pharmaceutical drug to be delivered which serves as an        intraocular implant. The capsule is inserted in the vitreous        region of the eye by making an incision in the eye, inserting        the capsule and closing the incision. The capsule remains in        place for a period of time and may be removed by making a second        surgical incision into the eye and retrieving the device. The        capsule has an attached tube which passes through the surface of        the eye and extends outward from the eye useful for the        subsequent injection of a drug. While in the vitreous, the        device is not anchored and may move about freely. Further, Zhou        et al. discloses a multiple-drug implant comprising        5-fluorouridine, triamcinolone, and human recombinant tissue        plasminogen activator for intraocular management of        proliferative vitreoretinopathy (PVR) (Zhou, T, et al. 1998,        “Development of a multiple-drug delivery implant for intraocular        management of proliferative vitreoretinopathy” J. Controlled        Release 55:281-295).

According to certain embodiments, compositions including agents areformulated and administered so as to apply a dosage effective foralleviating oxidative stress in the interior and posterior of the eye,and/or inhibiting the development of macular degeneration, otherretinopathies or uveitis in the eye, among other utilities as discussedherein. In general, it may be preferred that the active amount be fromabout 0.1% to about 10.0% weight by volume in the formulation. In someembodiments, it is preferable that the active drug concentration be0.25% to about 10.0% weight by volume. The concentration of thehydroxylamine component will preferably be in the range of about 0.1 μMto about 10 mM in the tissues and fluids. In some embodiments, the rangeis from 1 μm to 5 mM, in other embodiments the range is about 10 μM to2.5 mM. In other embodiments, the range is about 50 μM to 1 mM. Mostpreferably the range of hydroxylamine concentration will be from 1 to100 μM. The concentration of the reducing agent will be from 1 μM to 5mM in the tissues and fluids, preferably in the range of 10 μM to 2 mM.The concentrations of the components of the composition are adjustedappropriately to the route of administration, by typical pharmacokineticand dilution calculations, to achieve such local concentrations.

Other forms of administration, wherein the delivery to the eye is notcalled for, may include oral tablets, liquids and sprays; intravenous,intramuscular, intraarterial, subcutaneous and intraperitonealinjections; application to the skin as a patch or ointment; enemas,suppositories, or aerosols.

The compositions of the invention may contain at least one adjunctcompound in addition to an agent of the invention, wherein the adjunctcompound is useful for treating a disease or disorder that is the targetof the agent of the invention. Thus the compositions may include atherapeutic agent and an adjunct compound. The agent and compound(s) maybe administered sequentially or concurrently. Similarly, the methods ofthe invention include using such combination therapy.

For effective treatment of macular degeneration or any of the otherretinopathies or eye conditions described herein, one skilled in the artmay recommend a dosage schedule and dosage amount adequate for thesubject being treated. It may be preferred that dosing occur one to fourtimes daily for as long as needed. The dosing may occur less frequentlyif the compositions are formulated in sustained delivery vehicles, orare delivered via implant or intravitreal injection. The dosage schedulemay also vary depending on the active drug concentration, which maydepend on the hydroxylamine used and on the needs of the patient.

An ophthalmologist or one similarly skilled in the art will have avariety of means to monitor the effectiveness of the dosage scheme andadjust dosages accordingly. For example, effectiveness in the treatmentof macular degeneration or other retinopathies may be determined byimprovement of visual acuity and evaluation for abnormalities andgrading of stereoscopic color findus photographs. (Age-Related EyeDisease Study Research Group, NEI, NIH, AREDS Report No. 8, 2001, Arch.Ophthalmol. 119: 1417-1436). Effectiveness in the treatment of uveitismay be determined by improvement in visual acuity and vitreous haze andcontrol of inflammation (Foster et al., 2003, Arch. Ophthalmol. 121:437-40). Following such evaluation, the ophthalmologist may adjust thefrequency and/or concentration of the dose, if needed.

Ilustrative Examples

Methods:

Methods Summary: The CARD (caspase activation and recruitment domain)was amplified from the human apoptosis-associated speck-like proteincontaining a CARD (ASC) gene and fused to the cell-penetrating peptidesequence derived from the HIV Tat protein using PCR. The product wasfused to either a puromycin resistance gene in a lentiviral vectorplasmid or to a secretable form of GFP through a furin cleavage site.The sequence of the TatCARD insert was verified by DNA sequencing andcloned in an AAV plasmid. The monocytic cell line THP-1 and the retinapigmented ephitelium like cell line ARPE-19 were transduced withlentiviral vectors to generate stable cell lines by selecting forpuromycin resistant cells. Expression of TatCARD was determined bywestern blot, and its biological effect on LPS or 4-HNE inducedsecretion of IL-1β was measured by ELISA. Cellular localization of thesecretable TatCARD was determined by fluorescent microscopy. Detectionof secreted TatCARD was inferred western blot using cell conditionedmedia.

Cell Culture. The HEK293T cell line was grown in DMEM media supplementedwith 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin(Pen-Strep) solution. The ARPE-19 cell line was obtained from ATCC,which validated its identity. Cell stocks were frozen upon arrival andpassaged fewer than 4 times before being discarded. ARPE19 cells weregrown in DMEM/F12 (50/50) media supplemented with 10% FBS and 1%Pen-Strep. THP-1 cells were grown in RPMI-1640 supplemented with 10% FBSand 1% Pen-Strep. All the cell cultures were maintained in an incubatorat 37° C. with 5% CO₂. All stable cell lines generated by lentiviralvector transduction were grown in the corresponding media supplementedwith puromycin at a dose of 1 μg/mL.

Transfection. Cells were plated at 8×10⁵ cells per well in a 6-wellplate with complete growth medium and incubated for 24 hours. The nextday, complete growth medium was replaced in each well with 2 mL ofserum- and antibiotic-free medium. Plasmid DNA complexes were generatedby diluting 4 μg of the corresponding DNA in 100 μL of sterile phosphatebuffered saline (PBS) and 10 μg of a 1 μg/μL of polyethyleneimine²⁸(PEI) in 100 μL of PBS. Dilutions were incubated at room temperature for5 minutes. DNA:PEI complexes were made by mixing the diluted DNA and PEIand incubating them for 20 minutes at room temperature. Complexes wereoverlaid on the cells drop wise and cells were maintained at 37° C. for18 hours. The transfection was stopped by removing thecomplex-containing medium and replacing it with 3 mL of complete growthmedium. Cells were grown for another 24 hours at 37° C. Afterwards,cells were harvested by trypsin treatment and centrifugation.

Viral Vectors. All the lentiviral vectors were created using thepCDH-EF1-MCS-T2A-Puro plasmid (Systems Biosciences, Mountain ViewCalif.). The transgenes were cloned using the EcoRI and the NotIrestriction sites in the multiple cloning sites. Plasmids were grown inDH5α cells and sequenced by the Sanger method²⁹. To generate viralparticles, the plasmids were co-transfected with the pPACKH1 lentivectorpackaging kit (Systems Biosciences, Mountain View Calif.) in HEK293Tcells. The lentiviral vector containing media were harvested at 48 hoursafter the co-transfection and were centrifuged at 3,000 rpm for 5minutes at 4° C. These vector containing media were filtered using a0.22 μm syringe filter.

Immunoprecipitation. ARPE-19 cells expressing either TatCARD or only thepuromycin resistance gene were disrupted in NP-40 lysis buffer (1%NP-40, 150 mM NaCl, 50 mM Tris-Cl pH 8.0) supplemented with ProteaseInhibitors Cocktail (Thermo Fisher Scientific, Rockford Ill.) and 2 mMEDTA. Samples were kept on ice for 20 minutes and mixed by vortexingevery ten minutes followed by a centrifugation at 16,000×g for 15minutes at 4° C. Lysate was collected, and the protein concentration ofthe was measured with the DC Protein Assay (Bio-Rad, Hercules Calif.)according to the manufacturer's protocol. Lysates were diluted to 1μg/μl. A total of 500 μg of lysate was incubated with 1 μg of normalrabbit IgG antibody (Santa Cruz Biotechnology, Dallas Tex.) and 20 μLprotein A/G-agarose beads (Santa Cruz Biotechnology, Dallas Tex.) at 4°C. for 1 hour in a rotating mixer. Beads were pelleted by centrifugationat 1,000×g for 5 minutes at 4° C. A total of 5 μg of anti-Caspase-1antibody (Millipore) were added to 20 μg protein A/G-agarose beads in atotal volume of 500 μL PBS and were incubated at 4° C. for 1 hour in arotating mixer. Antibody/beads complex was pelleted by centrifugation asdone previously and was resuspended in 500 μL of 0.2M triethanolamine pH8.3 containing 20 mM of the cross-linking agent dimethyl pimelimidate(Sigma-Aldrich, St Louis Mo.). The complexes were incubated for 1 hourat room temperature in a rotating mixer. The cross-linking reaction wasquenched by adding 50 μL of 1M Tris-HCl pH 7.5 and incubating for 1 hourat room temperature in a rotating mixer. The complexes were pelleted bycentrifuging as done previously. The complexes were washed with 500 μLof 0.2 M glycine HCl pH 2.5 by incubating them for 1 minute at roomtemperature in a rotating mixer. These complexes were then washed 3times with PBS containing 0.01% Tween-20. A total of 500 μL of dilutedlysate (500 μg protein) was used to resuspend the pellet. Samples werekept at 4° C. overnight in a rotating mixer. The next day, samples werecentrifuged at 1,000×g for 5 minutes at 4° C. Supernatant was removedand the pellet was washed 4 times with NP-40 lysis buffer byresuspending and centrifuging as in previous steps. After the last wash,the pellet was resuspended in 60 μL of Laemmlli sample buffer and boiledfor 5 minutes. Samples were centrifuged at 16,000×g for 10 seconds, andsupernatant was transferred to a new 1.5 mL microcentrifuge tube. Atotal of 20 μL of sample were analyzed in a 12% SDS polyacrylamide geland transferred onto PVDF membranes using the iBlot system. Membraneswere probed with anti-Caspase-1 (Millipore, 1:1000 dilution) or anti-T2A(Millipore, 1:1000 dilution)

Enzyme Linked Immunosorbent Assay (ELISA). Medium was harvested from theindicated cultures and 100 μL aliquots were used to quantify IL-1βconcentration. The ELISA kit for the human IL-1β was purchased fromRayBiotech (Norcross, Ga.). The concentration of IL-1β was determinedaccording to the manufacturer's protocol.

Western Blot. Cells were disrupted as described above. Protein lysateswere diluted in Laemmli sample buffer containing 100 μM DTT and boiledfor 5 minutes. Equal amounts of protein were separated by SDSpolyacrylamide gel electrophoresis and transferred into a PVDF membraneusing the iBlot system (Invitrogen, Grand Island, N.Y.). This membranewas blocked with a proprietary blocking buffer from Li-Cor (Li-CorBiosciences, Lincoln, Ne.) for 1 hour at room temperature and incubatedovernight with the designated primary antibody at 4° C.

Endotoxin-Induced Uveitis (EIU) mouse model. Mice of the C57B/6 strainwere injected in the vitreous of each eye with 3×10⁹ vector genomes ofAAV2 expressing either GFP or sGFP-TatCARD from the CMV enhancer-chickenbeta actin (CBA) promoter. One month after the injection GFP expressionwas observed by fluorescent funduscopy. The next day, mice were injectedintravitreally in each eye with 25 ng of LPS. After 24 hours, these micewere sacrificed by inhalation with CO₂ followed by thoracotomy, andtheir eyes were enucleated and placed in 4% paraformaldehyde at 4° C.overnight. Eyes were embedded in paraffin were sectioned through thecornea-optic nerve axis at a thickness of 12 μm. The sections werecollected in independent slides with sections on the same slide having adifference of 96 μm. Slides were stained with hematoxylin and eosin tovisualize infiltrating cells. These cells were counted in images of thesections by two independent observers.

Fundoscopy. We used digital fundus imaging with a Micron III retinalimaging microscope (Phoenix Research Laboratories, Pleasaton, Calif.) tomonitor gene expression. Conscious mice had their eyes dilated with 1%atropine and 10% phenylephrine. Mice were then anesthetized with amixture of ketamine and xylazine in normal saline. To avoid loss ofmoisture from the ocular surface during the procedure mice received adrop of 2.5% hypermellose ophthalmic demulcent solution (Gonak, AKORN,Lake Forest, Ill.). B. Using the fluorescein filters we measure GFPfluorescence using the same exposure time for all the eyes.

Statistical Analysis. Statistical analysis was performed using theGraphpad Prism 5 software. Averages of replicate experiments werecompared by ANOVA followed by the Newman-Keuls test to detectdifferences among all groups. Statistical significance was reportedwhenever the calculated p-value was ≤0.05.

Results: The expression of the TatCARD significantly inhibited the LPSinduced secretion of IL-1β from THP-1 cells. The cellular distributionof sGFP-TatCARD in transfected cells was punctate in contrast to thecytoplasmic distribution of GFP. The fused sGFP-TatCARD construct wasdetected in tranfected cell lysates, whereas the cleaved GFP wasdetected in the corresponding cell conditioned media. Stable ARPE-19cells transduced with either empty lentivector control or TatCARDlentivector were stimulated with 4-HNE at 300 for 24 hours. The levelsof secreted IL-1β in the conditioned media were lower in cellsexpressing the TatCARD construct than in the empty lentivector controlcells.

Conclusions: The expression of TatCARD inhibits the LPS and the 4-HNEinduced secretion of IL-1β. We have successfully constructed asecretable form of the TatCARD protein that may be useful in blockingretinal and RPE inflammation.

Although more than one route can be used to administer a particularagent, a particular route can provide a more immediate and moreeffective reaction than another route. Accordingly, the described routesof administration are merely exemplary and are in no way limiting.

It should be borne in mind that all patents, patent applications, patentpublications, technical publications, scientific publications, and otherreferences referenced herein are hereby incorporated by reference inthis application in order to more fully describe the state of the art towhich the present invention pertains.

Reference to particular buffers, media, reagents, cells, cultureconditions and the like, or to some subclass of same, is not intended tobe limiting, but should be read to include all such related materialsthat one of ordinary skill in the art would recognize as being ofinterest or value in the particular context in which that discussion ispresented. For example, it is often possible to substitute one buffersystem or culture medium for another, such that a different but knownway is used to achieve the same goals as those to which the use of asuggested method, material or composition is directed.

It is important to an understanding of the present invention to notethat all technical and scientific terms used herein, unless definedherein, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. The techniques employed herein arealso those that are known to one of ordinary skill in the art, unlessstated otherwise. For purposes of more clearly facilitating anunderstanding the invention as disclosed and claimed herein, thefollowing definitions are provided.

While a number of embodiments of the present invention have been shownand described herein in the present context, such embodiments areprovided by way of example only, and not of limitation. Numerousvariations, changes and substitutions will occur to those of skill inthe art without materially departing from the invention herein. Forexample, the present invention need not be limited to best modedisclosed herein, since other applications can equally benefit from theteachings of the present invention. Also, in the claims,means-plus-function and step-plus-function clauses are intended to coverthe structures and acts, respectively, described herein as performingthe recited function and not only structural equivalents or actequivalents, but also equivalent structures or equivalent acts,respectively. Accordingly, all such modifications are intended to beincluded within the scope of this invention as defined in the followingclaims, in accordance with relevant law as to their interpretation.

While one or more embodiments of the present invention have been shownand described herein, such embodiments are provided by way of exampleonly. Variations, changes and substitutions may be made withoutdeparting from the invention herein. Accordingly, it is intended thatthe invention be limited only by the spirit and scope of the appendedclaims. The teachings of all references cited herein are incorporated intheir entirety to the extent not inconsistent with the teachings herein.

What is claimed is:
 1. A method for the amelioration or treatment of anocular disease or ocular condition in a subject, the method comprising:intravitreally administering a therapeutically effective amount of aviral vector comprising an expression construct comprising (a) anucleotide sequence encoding a fusion protein comprising acell-penetrating peptide derived from the HIV Tat protein (Tat) and aCaspase Activation and Recruitment Domain (CARD) and (b) a nucleotidesequence encoding a secretion signal peptide to the subject's eye,wherein the Tat comprises amino acids 1-9 of the amino acid sequence ofSEQ ID NO: 1 and the CARD comprises amino acids 10-100 of the amino acidsequence of SEQ ID NO: 1 wherein the ocular disease or ocular conditionis selected from the group consisting of ocular inflammation, maculardegeneration, age-related macular degeneration (AMD), geographicatrophy, wet AMD, dry AMD, drusen formation, dry eye, diabeticretinopathy, vitreoretinopathy, corneal inflammation, uveitis, ocularhypertension and glaucoma.
 2. The method of claim 1, wherein said viralvector is an AAV vector.
 3. The method of claim 1, wherein said viralvector is a retroviral vector, a lentiviral vector, an adenoviralvector, a Herpes viral vector, a Hepatitis viral vector, an SV40 vector,an EBV vector, an adeno-associated virus (AAV) vector or a nonviralvector.
 4. The method of claim 1, wherein said method comprises treatinga subject who exhibits one or more of the following symptoms: drusenformation, eye pain, eye redness, light sensitivity, tearing, or blurredvision.
 5. The method of claim 4, wherein said method comprises treatinga subject who exhibits two or more of the following symptoms: drusen,eye pain, eye redness, light sensitivity, tearing, or blurred vision. 6.The method of claim 4, wherein said subject exhibits drusen formation.7. The method of claim 2, wherein the AAV vector is an AAV2 vector. 8.The method of claim 1, wherein the secretion signal peptide is an IgKsignal peptide.